The Escherichia coli cAMP receptor protein (CRP) has been the model transcription factor for our understanding of ligand sensing, DNA recognition and transcriptional regulation. Proteins sequentially and structurally similar to CRP are widespread throughout the domain of the eubacteria and form the CRP-FNR superfamily (termed CRP homologs here). CRP homologs allow each bacterium to quickly adapt to environmental changes by sensing environmental cues and modulating the expression of the necessary genes, often on a global scale. The homologs found in bacterial pathogens serve as virulence factors, so the study of the family proteins is also of biomedical importance. In recent years, bacterial genomes have continuously been sequenced, which has generated more than 7,000 protein family members. The long-term goal of this study is to understand these unknown CRP homologs systematically and at the genome-wide level. This will reveal a wider functional scope of the superfamily, which includes pivotal virulence roles of some members in human disease. Unfortunately, conventional experimental approaches lack the means to characterize the overwhelming number of homologs within a practical time frame. While bioinformatics may provide us a more rapid estimate of homolog identity, it cannot provide the accurate set of information made available through experimental data. Intrinsic hurdles to studying any unknown CRP homologs originate from the two-pronged function (ligand binding and DNA binding) shared by the family members. If one wants to identify the ligand, one has to know the target DNA in advance for functional assay. Conversely, without knowing the ligand that activates the protein, one cannot confidently activate the protein, which is a prerequisite for identifying or evaluating the DNA targets. The specific goal of this proposal is to develop experimental tools bypassing the intrinsic problem to systematically characterize unstudied CRP homologs. Thus, the methodological core of this proposal lies in the use of various types of chimeric proteins that fuse a portion of the E. coli CRP to another portion of various unknown CRP homologs. Since the E. coli CRP is well characterized in each of the two domains (one for ligand binding and the other for DNA binding) is well characterized, the approach enables us to either activate the chimera (via cAMP, the ligand of CRP) or to functionally assay the protein (via the known target DNA of CRP). The idea is based on our successful precedents of chimeric proteins, and the approach, while not established, potentially provides an unmatched advantage for the functional elucidation of unknown CRP homologs. Then, the chimeric approach will be combined with other novel concepts and also with general methodologies. These methods will be systematically applied to several CRP homologs to test their feasibility to unknown CRP homologs. Specific aims are (i) developing new strategies for identifying the ligands of unknown CRP homologs, (ii) elucidating DNA-protein interaction rules for CRP homologs and (iii) applying gain-of-function strategies to CRP homologs for their genome-wide functions. In addition, the proposed experiments will provide an excellent research opportunity for graduate and undergraduate students in a minority-serving institution. PUBLIC HEALTH RELEVANCE: The CRP homologs found in bacterial pathogens such as Pseudomonas aeruginosa and Listeria monocytogenes act as virulence factors. This proposal will systematically characterize three P. aeruginosa CRP homologs of known or potential virulence factors. The pathogen is a frequent cause of nosocomial infections due to its natural and acquired resistance to many antibiotics. The proteins can be good therapeutic targets; therefore, the knowledge gained from this study might lead to the development of an alternative therapeutic strategy to control the pathogen.